The present invention relates to wound healing and also to regulating fibrosis in the treatment of conditions in which fibrosis is a major mechanism of tissue repair or where excessive fibrosis leads to pathological derangement and malfunctioning of tissue.
Wound healing in adults is a complicated reparative process. The healing process begins with the recruitment of a variety of specialised cells to the site of the wound and involves extracellular matrix and basement membrane deposition, angiogenesis, selective protease activity and re-epithelialisation. An important component of the healing process in adult mammals is the stimulation of fibroblasts to generate the extracellular matrix. This extracellular matrix constitutes a major component of the connective tissue which develops to repair the wound area.
The connective tissue that forms during the healing process is often fibrous in nature and commonly forms into a connective tissue scar (a process known as fibrosis).
A scar is an abnormal morphological structure resulting from a previous injury or wound (e.g. an incision, excision or trauma). Scars are composed of a connective tissue which is predominately a matrix of collagen types 1 and 3 and fibronectin. The scar may consist of collagen fibres in an abnormal organisation (as seen in scars of the skin) or it may be an abnormal accumulation of connective tissue (as seen in scars of the central nervous system). Most scars consist of abnormally organised collagen and also excess collagen. In man, in the skin, scars may be depressed below the surface or elevated above the surface of the skin. Hypertrophic scars are a more severe form of normal scarring, are elevated above the normal surface of the skin and contain excessive collagen arranged in an abnormal pattern. A keloid is another form of pathological scarring which is not only elevated above the surface of the skin but also extends beyond the boundaries of the original injury. In a keloid there is excessive connective tissue which is organised in an abnormal fashion predominately in whirls of collagenous tissue. There are genetic predispositions to forming both hypertrophic scars and keloids. They are particularly common in Africo-Carribean and Mongoloid races.
There is a need to provide medicaments that promote the healing of wounds. For example, it is often desirable to increase the rate of healing in the case of acute wounds (such as penetrative injuries, burns, nerve damage or even wounds resulting from elective surgery), chronic wounds (such as diabetic, venous and decubitus ulceration) or for generally healing compromised individuals (for example the elderly). In these examples, the wounds can severely influence quality of life or even result in death and therefore the rate of healing often needs to be increased as much as is clinically possible. Where the rate of wound healing is increased, there is often an associated increase in scar formation but this may be of secondary importance compared to the desired increase in the rate of healing.
The term xe2x80x9cwoundxe2x80x9d as used herein is exemplified but not limited to injuries to the skin. Other types of wound can involve damage, injury or trauma to an internal tissue or organ such as the lung, kidney, heart, gut, tendons or liver.
There are however other instances where the regulation of scar formation is of primary importance and the rate of wound healing is only of secondary consideration. Examples of such situations are scars of the skin where excessive scarring may be detrimental to tissue function and particularly when scar contracture occurs (for instance skin burns and wounds which impair flexibility of a joint). The reduction of scarring to the skin when cosmetic considerations are important is also highly desirable. In the skin, hypertrophic or keloid scars (particularly in Africo-Caribbean and Mongoloid races) can cause functional and cosmetic impairment and there is a need to prevent their occurrence. Scarring resulting from skin grafts in both donor sites and from the application of artificial skin can also be problematic and need to be minimised or prevented.
As well as scars of the skin, internal scarring or fibrosis can be highly detrimental and specific examples include:
(i) Within the central nervous system, glial scarring can prevent neuronal reconnection (e.g. following neuro-surgery or penetrating injuries of the brain).
(ii) Scarring in the eye can be detrimental. In the cornea, scarring can result in abnormal opacity and lead to problems with vision or even blindness. In the retina, scarring can cause buckling or retinal detachment and consequently blindness. Scarring following wound healing in operations to relieve pressure in glaucoma (e.g. glaucoma filtration surgery) results in the failure of the surgery whereby the aqueous humour fails to drain and hence the glaucoma returns.
(iii) Scarring in the heart (e.g. following surgery or myocardial infarction) can give rise to abnormal cardiac function.
(iv) Operations involving the abdomen or pelvis, often result in adhesion between viscera. For instance, adhesions between elements of the gut and the body wall may form and cause twisting in the bowel loop leading to ischaemia, gangrene and the necessity for emergency treatment (untreated they may even be fatal). Likewise, trauma or incisions to the guts can lead to scarring and scar contracture to strictures which cause occlusion of the lumen of the guts which again can be life threatening.
(v) Scarring in the pelvis in the region of the fallopian tubes can lead to infertility.
(vi) Scarring following injury to muscles can result in abnormal contraction and hence poor muscular function.
(vii) Scarring or fibrosis following injury to tendons and ligaments can result in serious loss of function.
Related to the above is the fact that there are a number of medical conditions known as fibrotic disorders in which excessive fibrosis leads to pathological derangement and malfunctioning of tissue. Fibrotic disorders are characterised by the accumulation of fibrous tissue (predominately collagens) in an abnormal fashion within the tissue. Accumulation of such fibrous tissues may result from a variety of disease processes. These diseases do not necessarily have to be caused by surgery, traumatic injury or wounding. Fibrotic disorders are usually chronic. Examples of fibrotic disorders include cirrhosis of the liver, liver fibrosis, glomerulonephritis, pulmonary fibrosis, sclerodermia, myocardial fibrosis, fibrosis following myocardial infarction, central nervous system fibrosis following a stroke or neuro-degenerative disorders (e.g. Alzheimer""s Disease), proliferative vitreoretinopathy (PVR) and arthritis. There is therefore also a need for medicaments which may be used for the treatment of such conditions by regulating (i.e. preventing, inhibiting, or reversing) fibrosis/scarring in these fibrotic disorders.
Whilst the above considerations mainly apply to conditions, disorders or diseases of man it will be appreciated that wound healing, scarring and fibrotic disorders can also be problematic in other animals, particularly veterinary or domestic animals (e.g. horses, cattle, dogs, cats etc). For instance abdominal wounds or adhesions are a major reason for having to put down horses (particularly race horses), as are tendon and ligament damage leading to scarring or fibrosis.
There have been several recent developments in the fields of wound healing, scarring and fibrotic disorders. Some of these developments revolve around the recent understanding that an array of cytokines and growth factors are intimately involved in the repair of tissues.
WO-A-92/17206 discloses the use of neutralising agents for fibrosis promoting growth factors that may be used to inhibit scar formation during wound healing. For instance, WO-A-92/17206 demonstrates that compositions which specifically inhibit the activity of Transforming Growth Factors xcex21 and xcex22 and Platelet Derived Growth Factor are particularly beneficial for reducing scar formation.
WO-A-93/19769 discloses the use of non-fibrotic growth factors, such as Transforming Growth Factors xcex23 which was surprisingly found to promote healing of a wound without inducing fibrosis.
GB-A-2,288,118 discloses the use of specific antibodies generated against growth factors that improve healing by potentiating the actions of said growth factors.
Another development involves the use of mannose-6-phosphate for use in treating fibrotic disorders associated with accumulation of extracellular matrix and with elevated levels of Transforming Growth Factors xcex21 or xcex22 (GB-A-2,265,310). Mannose-6-phosphate is believed to interfere with the conversion of latent forms of these Transforming Growth Factors into their active form.
Other compositions that influence growth factor efficacy and promote wound healing are disclosed in WO-A-95/26203.
Despite these advances there remains a need to continue to develop medicaments that may be used to modulate the healing of wounds.
According to a first aspect of the present invention, there is provided the use of a compound that influences the sex hormone system for the manufacture of a medicament for the treatment of wounds or fibrotic disorders.
According to a second aspect of the present invention, there is provided a method of treating wounds or fibrotic disorders comprising providing at the site of the wound or fibrotic disorder a therapeutically effective amount of a compound which influences the sex hormone system.
According to a third aspect of the present invention there is provided a healing composition which comprises an amount of a compound that influences the sex hormone system which is therapeutically effective for treating wounds or fibrotic disorders and a pharmaceutically acceptable vehicle.
By xe2x80x9csex hormone systemxe2x80x9d it is meant the endocrine system that influences gender, sexual development, fertility, secondary sexual characteristics and in females the menstrual cycle and pregnancy. Compounds which are useful are those which have an influence on this system. Examples of such compounds are endogenous hormones such as oestrogens, androgens, progesterone, chorionic gonadotrophin, follicle stimulating hormone and luteinising hormone as well as precursors thereof.
In accordance with the invention, the inventors have established that compounds which influence the sex hormone system may be used for the treatment of wounds and fibrotic disorders. Depending on the compound used, various regulating effects may be provided for the treatment of wounds or fibrotic disorders and these are discussed more fully below.
The invention has been based on our studies which have shown that the rate of wound healing declines with the age of subject in terms of re-epithelialisation, extracellular matrix and basement membrane deposition. We also noticed that aged females heal more quickly than aged males. In females this was associated with aa increased number of wound fibroblasts. Transforming Growth Factor xcex21 (TGF-xcex21) levels and increased proteolytic activity compared to aged males but decreased compared to young males and females. It was also noticed that in young males there is less scarring compared to that seen in young females which we associate with differences between TGF-xcex21 levels observed in the two sexes. Another difference between the sexes was that healing of wounds in females is generally associated with greater levels of elastin and angiogenesis than in males.
These findings led us to the realisation that sex hormones and other compounds which influence the sex hormone system have an influence on the rate and quality (extent of scar formation or fibrosis) of wound healing and also influence fibrous tissue deposition in fibrotic disorders. This hypothesis was examined and confirmed by assessing the effect of Hormone Replacement Therapy (HRT) on the rate and quality of wound healing in post-menopausal women. Women taking HRT of oestrogen alone or oestrogen and progesterone had significantly increased rates of skin wound healing (in terms of re-epithelialisation and extracellular matrix deposition) compared to age-matched women not on medication. Proteolytic activity both in normal (uninjured) skin and in the wounds of the post-menopausal women on HRT was reduced to that of a 20-30 year female age group. These effects were also associated with reversal of age related changes in the transforming growth factor xcex21 or interleukin-1 profiles in normal skin.
Although the inventors do not wish to be constrained by any hypothesis, they believe it is possible that the mechanism by which the sex hormones and other compounds which influence the sex hormone system exert their wound healing effect is by modulating the activity of molecules which regulate wound heating such as the cytokines (e.g. TGF-xcex21, Platelet Derived Growth Factor or interleukin-1) and thereby influence cellular function (for example the function of fibroblasts). For instance, our in vitro studies have established that oestrogen increases fibroblast TGF-xcex2 production which may be associated with the effect of the oestrogens to cause an increase in the rate of wound healing. Other compounds which influence the sex hormone system also modulate fibroblast activity. For example progesterone inhibits the proliferation of aged fibroblasts whereas androgens have similar effects to those of the oestrogens.
We have found that compounds which influence the sex hormone system also modulate enzyme profiles in a wound or tissue affected by a fibrotic disorder. In particular we have found that enzyme levels of matrix metalloproteinases (MMPs) (especially MMP2 and MMP9) as well as other lytic enzymes such as elastases are modulated by compounds such as oestrogen, progesterone and testosterone. We believe this modulation is sufficient to influence the rate of wound healing or to modulate fibrosis (and thereby influence scarring or fibrotic disorders) and it is possible that these effects represent a complementary, additional or even alternative mechanism (to that discussed in the preceding paragraph) by which compounds which influence the sex hormone system are able to treat wounds or fibrotic disorders.
Several classes of compound are capable of influencing the sex hormone system. Such compounds include hormones, hormone receptor agonists or antagonists, agents which modulate the release of endogenous activators or inhibitors of hormone receptors, agents which modulate the synthesis of endogenous hormone receptors ligand, agents which modulate the breakdown of endogenous hormone receptors ligand, agents which modulate hormone receptor expression or activity and agents which enhance the mechanisms involved in signal transduction between the receptor of the sex hormone system and effector systems.
Preferred compounds which influence the sex hormone system are hormones (or biologically active derivatives thereof) and agonists and antagonists of hormonal receptors. It is most preferred that the compound is a steroid sex hormone (such as oestrogen, progesterone or testosterone) or an agonist or antagonist of sex steroid hormonal receptors.
Compounds which influence the sex hormone system have been found, in accordance with the present invention, to regulate wound healing in one of two principal ways (depending on the particular compound used). These ways are described below with reference to the first and second embodiments of the invention.
In accordance with the first embodiment of the invention, we have established that certain compounds which influence the sex hormone system are capable of accelerating the rate at which a wound heals, although this may be at the expense of providing increased scarring or fibrosis. Such compounds are obviously particularly useful where speed of wound healing is a priority and the quality of any scar is a secondary consideration. Thus such compounds will be useful for acute wounds (such as penetrative injuries, burns, nerve damage, damaged ligaments or tendons, or even wounds resulting from elective surgery) and chronic wounds (such as diabetic, venous and decubitus ulceration). Such wounds can severely influence quality of life or even result in death and therefore the rate of healing may need to be increased as much as is clinically possible.
The compounds which are most effective in accordance with the first embodiment of the invention arc generally those which promote oestrogenic activity at the site of the wound. It is this promotion which accelerates the wound healing.
Examples of compounds which may be used to promote oestrogenic activity include oestrogens, oestrogen receptor agonists (such as ethinylyoestradiol, dienoestrol, mestranol, oestradiol, oestriol, conjugated oestrogens, piperazine oestrone sulphate, stilboestrol, fosfesterol tetrasodium, polyestradial phosphate, tibolone), inhibitors of oestrogen or oestrogen receptor agonist breakdown, phytoestrogens or even modulators of luteinising hormone, follicle stimulating hormone and chorionic gonadotrophin.
As an alternative to a promoter of oestrogenic activity, it is possible in accordance with the first embodiment of the invention, to use promoters of androgenic activity.
Preferred promoters of androgenic activity include androgen hormones (such as testosterone, dihydrotestosterone, 5xcex1-androstanediol), androgen receptor agonists (such as testosterone undecanoate, testosterone enanthate, testosterone esters, testosterone proprionate, mesterolone, danazol and gestrinone), inhibitors of androgen or androgen receptor agonist breakdown (such as aminoglutethamide), modulators of luteinising hormone and follicle stimulating hormone, anabolic steroids (such as nandrolone or stanozolol).
The preferred compound for use in the first embodiment of the invention is an oestrogen hormone receptor agonist. 17 xcex2-oestradiol is particularly preferred.
In accordance with the second embodiment of the invention we have established that certain compounds which are capable of influencing the sex hormone system are capable of regulating wound healing or fibrotic disorders by improving the quality of scar formation or preventing inappropriate fibrosis, although possibly at the expense of the rate of wound healing.
Thus such compounds (which inhibit fibrosis) used according to the second embodiment of the invention are useful in situations or conditions where scarring needs to be prevented or reduced such as:
(i) where scars of the skin may be excessive and/or detrimental to tissue function and particularly when scar contracture occurs or may occur (for instance skin burns and wounds which impair flexibility of a joint and particularly scarring in children);
(ii) scarring to the skin when cosmetic considerations are important;
(iii) when hypertrophic or keloid scars (particularly in Africo-Caribbean and Mongoloid races) may occur which can cause functional and cosmetic impairment;
(iv) scarring resulting from skin grafts in both donor sites and from the application of artificial skin;
(v) scarring within the central nervous system (e.g. following neuro-surgery or penetrating injuries of the brain), for example glial scarring can prevent reconnection of severed neurons;
(vi) scarring in the eye and particularly of the cornea (scarring can result in abnormal opacity and lead to problems with vision or even blindness), in the retina (scarring can cause buckling or retinal detachment and consequently blindness) and scarring following wound healing in operations to relieve pressure in glaucoma (e.g. glaucoma filtration surgery) which can result in the failure of the surgery whereby the aqueous humour fails to drain and hence the glaucoma returns;
(vii) scarring in the heart (e.g. following surgery or myocardial infarction) which can give rise to abnormal cardiac function;
(viii) scarring of the gut such as may occur following operations involving the abdomen or pelvis that result in adhesion between viscera (adhesions between elements of the gut and the body wall can form and cause twisting in the bowel loop leading to ischaemia, gangrene and the necessity for emergency treatmentxe2x80x94untreated they may even be fatal); likewise, trauma or incisions to the guts can lead to scarring and scar contracture or strictures which cause occlusion of the lumen of the guts which again can be life threatening;
(ix) scarring in the pelvis in the region of the fallopian tubes which can lead to infertility;
(x) scarring following injury to muscles which can result in abnormal contraction and hence poor muscular function;
(xi) scarring or fibrosis following injury to tendons and ligaments which can result in serious loss of function.
Compounds (which inhibit fibrosis) used according to the second embodiment of the invention are also useful for the treatment or prevention of fibrotic disorders such as cirrhosis of the liver, liver fibrosis, glomerulonephritis, pulmonary fibrosis, scleroderma, myocardial hibernation, fibrosis following myocardial infarction, central nervous system fibrosis following a stroke or neuro-degenerative disorders (e.g. Alzheimer""s Disease), proliferative vitreoretinopathy (PVR) and arthritis.
Examples of compounds for use in accordance with the second embodiment of the invention include promoters of progesterone activity such as progesterone and other progesterone receptor agonists (such as allyoestrenol, desogestrel, dydrogesterone, ethynodiol diacetate, gestodene, gestranol hexatioate, hydroxyprogesterone hexanoate, levonorgestrel, megestrol acetate, medroxyprogesterone acetate, norethisterone, norethisterone acetate, norethisterone enanthate, norgestimate or norgesterel), inhibitors of progesterone or progesterone receptor agonist breakdown and modulators of luteinising hormone and/or follicle stimulating hormone.
Alternatively, the compound used in the second embodiment of the invention may be an inhibitor of oestrogenic activity. Preferred inhibitors of oestrogenic activity include oestrogen receptor antagonists (such as tamoxifen, clomiphene citrate or cyclofenil), inhibitors of oestrogen production (such as anastrozole, 4-hydroxy androstenedione, exemestane, oestrone-3-O-sulphate, fadrazole hydrochloride or formestane) and phytoestrogens. Tamoxifen is particularly useful for use according to the second embodiment of the invention.
As a further possibility, the compound used in the second embodiment of the invention may be an inhibitor of androgenic activity. Preferred inhibitors of androgenic activity include androgen receptor antagonists (such as cyproterone acetate or flutamide) and inhibitors of androgen production.
Other compounds which influence the sex hormone system may be used according to either the first or second embodiments of the invention. For instance precursors of sex hormones which are subsequently converted to the active compound may be used. Dehydroepiandrosterone (DHEA) and its sulphate ester. DHEA sulphate (DHEAS) and analogues thereof are precursors of oestrogens and androgens and may be used as compounds which influence the sex hormone system according to the first embodiment of the invention to promote the rate of wound healing. We were lead to investigating how efficacious DHEA and DHEAS and analogues thereof may be for modulating wound healing after observing that circulating levels of DHEA and DHEAS decline progressively and markedly with age (which we in turn correlated with slower wound healing), in distinct contrast to other steroids such as glucocorticoids, whose serum levels remain relatively well-preserved with age. We have conducted studies that demonstrate that supplementation of individuals with DHEA (as a precursor of androgens and oestrogen), influences wound healing in the aged by stimulating the rate of wound repair. Therefore DHEA or DHEAS and analogues thereof may be used according to the first embodiment of the invention to promote wound healing.
It will be appreciated that combinations of the abovedescribed compounds may be used to maximise efficacy of the medicament, For instance, a progesterone receptor agonist and an oestrogen receptor antagonist may be combined to maximise the effect on scarring and/or a fibrotic condition.
Other preferred combinations are ones that are effective for treating wounds such that the rate of healing is increased and fibrosis is also inhibited. A combination of an oestrogen receptor agonist and a progesterone receptor agonist may be used for this purpose.
Alternatively compounds of the invention may be given sequentially. For instance an oestrogen agonist may be given pre-operatively (or peri-operatively) to promote the healing of a subsequent surgical incision. Later, while a patient is recovering from the surgery, a progesterone agonist may be given to reduce scarring.
Preferred treatment regimes contemplated by the present invention are non-systemic treatments since systemic application of compounds influencing the sex endocrine system may have undesirable effects, such as influencing secondary sexual characteristics whereas non-systemic administration (e.g. topical application to the skin) only has a local action and therefore does not have such undesirable effects. However, there are cases (for example severe injury or when acute therapy is required) where systemic applications are useful.
The compositions of the invention may take a number of different forms depending, in particular on the manner in which the composition is to be used. Thus, for example, the composition may be in the form of a liquid, ointment, cream, gel, hydrogel, powder or aerosol, It will be appreciated that the vehicle of the composition of the invention should be one which is well tolerated by the patient and allows release of the active compound to the wound. Such a vehicle is preferably biodegradeable, bioresolveable and/or non-inflammatory.
When the compound is a steroid (such as the oestrogens, progesterone, androgens or DHEA) the vehicle may contain a carrier molecule which improves the aqueous solubility of the compound. A suitable carrier is 2-hydroxypropyl-xcex2-cyclodextrin which is preferably present in the composition in approximately equimolar concentrations to that of the steroid.
The composition of the invention may be used in a number of ways. Thus, for example, a composition in accordance with the first or second embodiment of the invention may be applied in and/or around a wound of a patient to provide the desired regulation of wound healing.
If the composition is to be applied directly to an actual wound, trauma or injury, then the pharmaceutically acceptable vehicle will be one which does not cause an inflammatory response or is toxic to the tissue.
It is however also possible to use compositions in accordance with the invention as a prophylactic, For instance, prior to surgery (particularly elective surgery) it may be desirable to provide a compound which influences the sex hormone system for regulation of healing of the subsequently formed surgical wound so as to increase the rate of wound healing (according to the first embodiment of the invention) or to reduce scarring and/or tract a fibrotic disorder (according to the second embodiment of the invention) as appropriate. In this case the vehicle of the composition will need to be one capable of delivering the compound to the target site. For example the vehicle may need to be suitable for carrying the compound across the keratinous layer of the skin. Examples of suitable vehicles for this purpose include dimethyl sulphoxide and acetic acid.
Composition may be provided on a sterile dressing or patch which may be used to cover or even pack a wound to be treated. In this respect conventional Hormone Replacement Therapy patches may be suitably used for treating wounds and/or fibrotic disorders.
A further important application of the composition of the invention relates to wound healing in the eye. For example, compositions in accordance with the first embodiment of the invention may be used to provide scarring (as well as increase the rate of healing) between the sclera and retina when it is desired to repair a tear in the latter. In this case, the composition of the invention may be an injectable solution. Alternatively, a composition in accordance with the second embodiment of the invention may be used to reduce or control scarring resulting from surgical operations on the eye, e.g. laser surgery on the cornea. In this case, the composition of the invention may be in the form of an eye drop.
Compositions in accordance with the invention may be used in a range of internal wound healing applications (in addition to that mentioned above for the eye). Thus for example, the composition may be formulated for inhalation for use in wound healing of the lungs or for the prevention or treatment of fibrosis and strictures in the lung.
It will be appreciated that the amount of compound that influences the sex hormone endocrine system to be incorporated in a composition in accordance with the invention and/or the amount of the compound to be applied to the wound site depends on a number of factors such as the biological activity and bioavailability of the compound, which in turn depends on the mode of administration and the physicochemical properties of the compound, Other factors include:
A) The half-life of the compound in the subject being treated.
B) The specific condition to be treated.
C) Whether quick healing or reduced scarring is desired.
D) The age of the subject.
E) The sex of the subject.
The frequency of administration will also be influenced by the above mentioned factors and particularly the half-life of the compound within the subject being treated.
Generally when the compositions are used to treat existing wounds or fibrotic disorders the compound should be administered as soon as the wound has occurred or the disorder has been diagnosed. Therapy with the composition should continue until the wound has healed to a clinicians satisfaction or, in the case of a fibrotic disorder, the risk or cause of abnormal fibrous tissue formation has been removed.
Compositions which promote the rate of wound healing according to the first embodiment of the invention should he applied to a wound as soon as possible after the wound has formed. For acute wounds and wounds of subjects who are healing competent (e.g. the young) application of the composition will ideally be at the time of wounding, preferably within hours of wounding and no longer than a few days post-wounding. For chronic wounds or wounds in the healing compromised (e.g. the elderly) administration should be as soon as possible.
Compositions which modulate scarring and/or fibrotic disorders according to the second embodiment of the invention should also be applied to a wound as soon as possible after the wound has formed. However fibrosis can develop over days or even weeks. Therefore the subject being treated may well benefit by administration of a compound (such as progesterone or tamoxifen) even if the composition is administered days or even weeks after the wound occurred or the disorder developed (or was diagnosed).
When used as a prophylactic (e.g. before surgery or when there is a risk of developing a fibrotic disorder) the compositions should be administered as soon as the risk of undesirable fibrosis or a potential for a poor rate of wound healing has been recognised (as may be the case in elderly subjects). For instance, a cream or ointment containing 17 xcex2-oestradiol may be applied to a site on the skin of subject where elective surgery is to be performed and an increased rate of wound healing is subsequently desired. In this case, the composition may be applied during the preoperative preparation of the subject or it may even be desirable to apply the composition in the hours or days preceding the surgery (depending upon the health status and age of subject as well as the size of the wound to be formed).
Frequency of administration will depend upon the biological half-life of the compound used. Typically a cream or ointment containing a compound should be administered to a target tissue such that the concentration of the compound at the wound site or tissue affected by a fibrotic disorder is maintained at a level suitable for having a therapeutic effect. This may require administration daily or even several times daily. In the case of the use of 17 xcex2-oestradiol for wound healing, we have found that administration of the compound (by a patch applied to the wound) for 24 hours post-wounding is sufficient to improve the rate at which the wound heals.
Known procedures, such as those conventionally employed by the pharmaceutical industry (e.g. in vivo experimentation, clinical trials etc), may be used to establish specific formulations of compositions and precise therapeutic regimes (such as daily doses of the compounds and the frequency of administration).
Generally, compositions in accordance with the invention will contain 0.001% to 4% by weight of the compound which influences the sex hormone system. Purely by way of example a composition containing between 0.005% and 1% by weight of oestriol, oestradiol, ethinyloestradiol or testosterone is suitable for application to an existing (i.e. xe2x80x9copenxe2x80x9d) wound.
By way of further example, a composition which is to be used preoperatively as a prophylactic may contain between 0.01% and 2% by weight of oestriol, oestradiol, ethinyloestradiol or testosterone to have the desired effect on wound healing.
A preferred composition for use in the present invention comprises a maximum of 1% (e.g. between 0.005% and 1%) to 17 xcex2-Oestradiol.
A suitable daily dose of a compound which influences the sex hormone system will depend upon the factors discussed above as well as upon the size of the wound to be treated. Typically the amount of a compound required for the treatment of wounds or fibrotic disorders will be within the range of 1 ng to 100 g of the active compound/24 hours depending upon the size of the wound or extent of fibrosis amongst several other factors. By way of example 0.5-500 xcexcg/24 hrs of 17 xcex2-oestradiol is a suitable dose for treating a wound (to increase the rate of healing) made by a 4 mm punch biopsy of the skin, more preferably 10-100 xcexcg/24 hrs of 17 xcex2-oestradiol is used and most preferably 25 xcexcg/24 hrs of 17 xcex2-oestradiol is used.
A preferred means of using protein or peptide compounds which influence the sex hormone system is to deliver the compound to the wound by means of gene therapy. For instance gene therapy could be used to increase expression of peptide ligands for chorionic gonadotrophin receptors, follicle stimulating hormone receptors or luteinising hormone receptors. Alternatively gene therapy may be used to modulate the expression of an enzyme involved in the synthesis of steroid sex hormones (e.g. oestrogens, androgens or progesterone). Therefore according to a fourth aspect of the present invention there is provided a delivery system for use in a gene therapy technique, said delivery system comprising a DNA molecule encoding for a protein which directly or indirectly modulates wound healing and/or modulates fibrosis or scarring by influencing the sex hormone system, said DNA molecule being capable of being transcribed to lead to the expression of said protein.
According to a fifth aspect of the present invention there is provided the use of a delivery system as defined in the preceding paragraph for use in the manufacture of a medicament for use in wound healing and/or modulation of fibrosis or scarring.
According to a sixth aspect of the present invention there is provided a method of treating a wound and/or modulating fibrosis or scarring comprising administering to a patient in need of treatment a therapeutically effective amount of a delivery system as defined for the fourth aspect of the invention.
The delivery systems are highly suitable for achieving sustained levels of an active agent at a wound site or site of fibrosis over a longer period of time than is possible for most conventional delivery systems. Protein may be continuously expressed from cells at the wound site or site of fibrosis that have been transformed with the DNA molecule of the fourth aspect of the invention. Therefore, even if the protein has a very short half-life as an agent in vivo, therapeutically effective amounts of the protein may be continuously expressed from the treated tissue.
Furthermore, the delivery system of the invention may be used to provide the DNA molecule (and thereby the protein which is an active therapeutic agent) without the need to use conventional pharmaceutical vehicles such as those required in ointments or creams that are contacted with the wound. This is particularly beneficial as it can often be difficult to provide a satisfactory vehicle for a compound for use in wound healing (which are required to be non-inflammatory, biocompatible, bioresorbable and must not degrade or inactivate the active agent (in storage or in use)).
The delivery system is such that the DNA molecule is capable of being expressed (when the delivery system is administered to a patient) to produce a protein which directly or indirectly has activity for wound healing and/or treatment of fibrosis or scarring. By xe2x80x9cdirectlyxe2x80x9d we mean that the product of gene expression per se has the required activity for wound healing and/or regulating fibrosis or scarring. By xe2x80x9cindirectlyxe2x80x9d we mean that the product of gene expression undergoes or mediates (e.g. as an enzyme) at least one further reaction to provide an agent effective for wound healing and/or regulating fibrosis or scarring.
The DNA molecule may be contained within a suitable vector to form a recombinant vector. The vector may for example be a plasmid, cosmid or phage. Such recombinant vectors are highly useful in the delivery systems of the invention for transforming cells with the DNA molecule.
Recombinant vectors may also include other functional elements. For instance, recombinant vectors can be designed such that the vector will autonomously replicate in the nucleus of the cell. In this case, elements which induce DNA replication may be required in the recombinant vector. Alternatively the recombinant vector may be designed such that the vector and recombinant DNA molecule integrates into the genome of a cell. In this case DNA sequences which favour targeted integration (e.g. by homologous recombination) are desirable. Recombinant vectors may also have DNA coding for genes that may be used as selectable markers in the cloning process.
The recombinant vector may also further comprise a promoter or regulator to control expression of the gene as required.
The DNA molecule may (but not necessarily) be one which becomes incorporated in the DNA of cells of the subject being treated. Undifferentiated cells may be stably transformed leading to the production of genetically modified daughter cells (in which case regulation of expression in the subject may be required e.g. with specific transcription factors or gene activators). Alternatively, the delivery system may be designed to favour unstable or transient transformation of differentiated cells in the subject being treated. When this is the case, regulation of expression may be less important because expression of the DNA molecule will stop when the transformed cells die or stop expressing the protein (ideally when the wound, fibrosis or scarring has been treated or prevented).
The delivery system may provide the DNA molecule to the subject without it being incorporated in a vector. For instance, the DNA molecule may be incorporated within a liposome or virus particle. Alternatively the xe2x80x9cnakedxe2x80x9d DNA molecule may be inserted into a subject""s cells by a suitable means e.g. direct endocytotic uptake.
The DNA molecule may be transferred to the cells of a subject to be treated by transfection, infection, microinjection, cell fusion, protoplast fusion or ballistic bombardment. For example, transfer may be by ballistic transfection with coated gold particles, liposomes containing the DNA molecule, viral vectors (e.g. adenovirus) and means of providing direct DNA uptake (e.g. endocytosis) by application of plasmid DNA directly to the wounded area topically or by injection.
The protein expressed from the DNA molecule may be one which directly or indirectly provides for wound healing with reduced scarring, one which provides an increase in the rate of wound healing whilst possibly resulting in increased scar formation or one which serves to regulate (inhibit, prevent or reverse) fibrosis.
Whilst the above considerations mainly apply to wounds and fibrotic disorders of man it will be appreciated that wound healing, scarring and fibrosis can also be problematic in other animals (especially domestic animals such as horses, dogs, cats etc). For instance abdominal wounds or adhesions are a major reason for having to put down horses, as are tendon and ligament damage leading to scarring or fibrosis. The compounds, compositions and delivery systems discussed above are suitable for use in the healing of such animals.